Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

Abstract

Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

Keywords: M. tuberculosis; phosphoproteomics; serine phosphorylation; threonine phosphorylation; tyrosine phosphorylation.

Figure 1

Growth curves of hyper-virulent M. tuberculosis Beijing strain. Growth monitored over a 24 day period using OD₆₀₀ measurements in 7H9 Middlebrooks liquid media supplemented with dextrose and catalase.

Figure 2

Functional classification of phosphorylated proteins. Percentage phosphorylated proteins per functional category were classified according to Tuberculist. Number of phosphorylated proteins identified in each functional category depicted above the black bars.